LWT - Food Science and Technology 187 (2023) 115304 Contents lists available at ScienceDirect LWT journal homepage: www.elsevier.com/locate/lwt Encapsulating capacity of ultra-high-pressure homogenization (UHPH): Replacement of milk fat by vegetable oils using buttermilk as a functional ingredient in yogurt processing Fatemeh Aghababaei , Antonio J. Trujillo , Bibiana Juan , Marta Capellas , Victoria Ferragut * Centre d’Innovació, Recerca i Transferència en Tecnologia dels Aliments (CIRTTA), TECNIO-UAB, MALTA Consolider Team, Departament de Ciència Animal i dels Aliments, Universitat Autònoma de Barcelona, UAB-Campus, 08193, Bellaterra, Spain A R T I C L E I N F O A B S T R A C T Keywords: This study investigated the characteristics of yogurts produced by substituting dairy fat with polyunsaturated Yogurt fatty acids (PUFA)-rich oils encapsulated with buttermilk (BM) in spray-dried emulsions (SDE). Two homoge­ Polyunsaturated fatty acids nization methods, conventional (CH) and ultra-high-pressure homogenization (UHPH), were compared to obtain Buttermilk the emulsions for spray drying. Recombined milks (RMs) were formulated using two different concentrations (4 Ultra-high-pressure homogenization Encapsulation g/100 g and 6 g/100 g) of SDE, followed by fermentation. Yogurt characteristics were evaluated during cold storage using various parameters, including coagulation properties, texture and rheology, microstructure, physicochemical characteristics (color, pH, total acidity, and water holding capacity), oxidative stability, main fatty acid profile, microbial assessment, and sensory evaluation. During cold storage, several parameters significantly influenced the yogurt characteristics. The CH yogurts exhibited higher textural parameters (firm­ ness and consistency) and viscoelastic parameters (G′ and G’’) compared to the UHPH yogurts at the same SDE concentration. However, UHPH yogurts generally showed better water holding capacity (WHC) values. UHPH yogurts also demonstrated superior stability to oxidation and higher PUFA content. The observed differences between the CH and UHPH treatments can be attributed to the structuring of fat-protein-BM into colloidal particles based on the homogenization system employed in this study. Neither of the homogenization systems nor the SDE content impacted yogurt flavor. 1. Introduction strategies have been extensively developed to protect PUFA from oxidation during processing and storage, focusing on the emulsification Functional foods have gained significant popularity in international system and compounds employed to shield them. Various compounds, markets over the past two decades due to their diverse health promoting such as proteins, carbohydrates, and low molecular weight emulsifiers benefits (Liu et al., 2019). Omega-3 fatty acids (FA) are essential for are commonly used to create stable emulsions for further drying and various biochemical processes, cell membranes, brain development, and encapsulation of oil droplets into microcapsules (Ruiz, Ortiz, & Segura, physiological functions in the human body. The global market for 2017). omega-3 FA supplements reached a value of USD 5,580,000 in 2020, Buttermilk (BM), a co-product of butter manufacturing, is an inex­ with an expected annual growth rate of 8.6% from 2020 to 2028 pensive and readily available ingredient in the food industry. It pos­ (Grandview Research, 2020). sesses emulsifying capacity and functionality, attributed to the polar To meet the demand for fats and oils rich in polyunsaturated FA lipids and proteins present in the milk fat globule membrane (MFGM) (PUFA), fortification of commonly consumed foods with these fatty (Barry, Dinan, & Kelly, 2017). These compounds have also been asso­ acids has become prevalent in the food industry. However, during pro­ ciated with notable health promoting benefits, including improved brain cessing, distribution, and handling, PUFA are susceptible to oxidation, cognitive development and immune system support for optimal growth resulting in off-flavor and a decrease in quality and health promoting in infants (Hernell, Timby, Domellöf, & Lönnerdal, 2016; Singh & Gal­ benefits (Uluata, McClements, & Decker, 2015). Encapsulation lier, 2017). Moreover, commercial BM has demonstrated excellent * Corresponding author. E-mail address: victoria.ferragut@uab.cat (V. Ferragut). https://doi.org/10.1016/j.lwt.2023.115304 Received 9 June 2023; Received in revised form 7 September 2023; Accepted 13 September 2023 Available online 15 September 2023 0023-6438/© 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). F. Aghababaei et al. LWT 187 (2023) 115304 encapsulating properties, surpassing skim milk powder when used to 2.2. Emulsion and yogurt preparation encapsulate fish oil (Augustin et al., 2015). It also mimics the natural milk fat globule membrane due to its polar lipid composition, potentially Preparation of emulsion and spray-dried emulsion (SDE) were pre­ enhancing the digestion and utilization of encapsulated omega-3 FA viously and fully described (Aghababaei, Cano-Sarabia, Trujillo, Que­ (Zhang et al., 2020). vedo, & Ferragut, 2021). The SDE composition used for yogurt The successful encapsulation of lipophilic compounds, such as PUFA- manufacturing was: 7 g/100 g BM, 30 g/100 g maltodextrin (MD), 10 rich oils, requires the formation of stable emulsions prior to dehydra­ g/100 g oil, 47 g/100 g total solids, and 3 g/100 g moisture. Four tion. Emulsions are thermodynamically unstable colloidal systems, different recombined milks (RMs) to produce yogurts were prepared by prone to creaming, aggregation, and coalescence. High-energy me­ varying the percentage of SDE added as well as the type of homogeni­ chanical devices such as high-pressure homogenizers or ultrasonic zation used to obtain the SDE. Thus, the nomenclature of yogurts in­ generators are commonly employed in the industry to produce stable dicates these two variables as follows: CH4 (4 g/100 g SDE treated by emulsions by reducing droplet size (Floury, Desrumaux, & Lardières, CH); CH6 (6 g/100 g SDE treated by CH); UH4 (4 g/100 g SDE treated by 2000; Weiss, Takhistov, & Julian, 2006). UHPH); and UH6 (6 g/100 g SDE treated by UHPH). To prepare the RM Ultra-high-pressure homogenization (UHPH) is a versatile technol­ for further preparation of yogurts, UHT skim milk was mixed with 3 g/g ogy operating at pressures up to 350 MPa, compared to conventional SMP and 4 or 6 g/100 g SDE. RMs were stirred with a blade stirrer for 10 homogenization (CH) typically ranging between 20 and 50 MPa. UHPH min and were further pasteurized in batch at 82 ◦ C for 20 min. After can inactivate microorganisms and enzymes, produce submicron emul­ cooling of the mixes, they were stored at 4 ◦ C overnight to complete sions with excellent physical stability, and induce changes in colloidal powder hydration. The next day, RMs were heated to 45 ◦ C, inoculated structures. The composition of emulsifying agents used in UHPH can with 0.02 g/100 g of the starter culture Y0-MIX 300 LYO 10 DCU influence the restructuring of the protective layer of droplets, impacting (Danisco, Buxières, France) containing Lactobacillus delbrueckii subsp. the techno-functional properties (Dumay et al., 2013; Sato, Matsumiya, bulgaricus and Streptococcus thermophilus, and incubated at 43 ◦ C until Kaneko, Okazaki, & Matsumura, 2021). pH 4.6 was reached. Yogurts were stored at 4 ◦ C for 24 h and further Spray drying is a cost-effective method widely used in the food in­ stirred with a Jata FP500P rod blender (Tudela, Navarra, Spain) for 1 dustry to convert emulsions into easy-to-handle, transport, and to pre­ min clockwise to breakdown the gels. Then, stirred yogurts were serve powder form. Encapsulation through spray drying enables the transferred to sterile media bottles and stored at 4 ◦ C until analysis. production of small-size particles resistant to chemical and physical damage, ensuring protection against oxidation, light, and temperature 2.3. Evaluation of coagulation properties (Di Giorgio, Salgado, & Mauri, 2019; Geranpour, Assadpour, & Jafari, 2020). The coagulation process was monitored on inoculated RM (10 mL) at Yogurt, a popular dairy product, is known for its natural composition 43 ± 2 ◦ C using an Optigraph System (Ysebaert Dairy Division, Frépil­ rich in nutrients and numerous health promoting benefits attributed to lon, France) which is a measure based on the attenuation of near- fermentation (Donovan & Rao, 2019; Gumus & Gharibzahedi, 2021; Kok infrared region (NIR) signal. From the coagulation curves (optical & Hutkins, 2018). It is an ideal candidate for nutrient fortification. signal vs time), three parameters were obtained (Fig. 1): onset of gela­ Substituting milk fat with PUFA-rich oils emulsified with MFGM com­ tion (OG) obtained from the maximum of the second derivative; ag­ ponents from buttermilk presents an opportunity to develop a balanced gregation rate (AR), calculated from the slope of the linear part of the functional food. The production of spray-dried emulsions (SDE) con­ curve; and final gel firmness (FGF) at the end of the coagulation, ob­ taining PUFA-rich oils stabilized by UHPH has been recently investi­ tained by the difference between the final and initial values of the op­ gated (Varela et al., 2022), demonstrating superior properties compared tical signals. to SDE produced by CH. This study aims to evaluate the influence of The acidification curves of RM during the fermentation process were homogenization type (CH and UHPH) used to produce SDE and the performed with a Cinac32 equipment (Ysebaert Dairy Division, Frépil­ concentration of SDE added to stirred yogurts on the relevant quality lon, France), by continuous monitoring of pH. Inoculated RMs (100 mL) characteristics of the final product. were poured into 250 mL Erlenmeyer flasks at 43 ◦ C in a water bath until yogurts reached pH 4.6. 2. Material and methods 2.1. Materials 2.4. Texture, rheology, and microstructure Materials used for yogurt manufacturing, UHT skim milk, and skim Textural characteristics were evaluated by using a Texture Analyzer milk powder (SMP), were purchased from a local supermarket. Ac­ TA.TX2 (Stable Micro Systems, Surrey, UK) equipped with a flat cylin­ cording to the supplier, SMP contained 32.5 g/100 mL protein, 1 g/100 drical probe 35 mm diameter, applying a constant speed of 1 mm/s up to mL fat, and 54.5 g/100 mL carbohydrate. Lyophilized yogurt culture, a depth of 5 mm. A back-extrusion test was performed to determine composed of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus Y0-MIX 300 LYO 10 DCU, was purchased from Danisco (Buxières, France). Refined sunflower was purchased from Gustav Heess Company (Barcelona, Spain). The characteristics and composition ac­ cording to the producer were: acid value = 0.1 g/100 mL (mg KOH/g); peroxide value (meq O2/kg) = 0.02, fatty acid composition (15–85 g/ 100 mL C-18:1, 50–72 g/100 mL C-18:2). Crude Chia oil was as follows: acid value = 0.27 g/100 mL (mg KOH/g), peroxide value = 1.5 (meq O2/kg). Fatty acid composition (6.35 g/100 mL C-18:1, 19.29 g/100 mL C-18:2, 64.85 g/100 mL C-18:3) according to the specifications was obtained from Interfat Natural Oils (Barcelona, Spain). All other chem­ icals used were of analytical or better grade. Fig. 1. Coagulation curves as a function of time. CT is the time at the maximum of first derivative value (dotted line). AR is the aggregation rate, which is the slope of the plot over the coagulation period. Final gelation firmness (FGF) was calculated as D1–D0. 2 F. Aghababaei et al. LWT 187 (2023) 115304 firmness (maximum force in compression) and consistency (area under procedure reported by Bondia-Pons, Moltó-Puigmartí, Castellote, and the compression curve) (Liu, Xu, & Guo, 2007). Briefly, 60 g of yogurt at López-Sabater (2007) was used with a slight modification. Gas chro­ 4 ◦ C was poured into an 80 mm height, 45 mm diameter plastic matography conditions were as follows: separation of fatty acid methyl container. The analysis was conducted in yogurts at the refrigeration esters (FAMEs) was carried out, the carrier gas was helium with a head temperature, about 4 ◦ C. pressure of 220 kPa, the split ratio was 50:1 and the injection volume Dynamic oscillatory testing of stirred yogurts was performed using a were 1 μL. The oven and detector temperature were 250 ◦ C and 260 ◦ C, rheometer Thermo Haake RS1 (Thermo Electron Corporation, Karls­ respectively. The temperature program was as follows: initial tempera­ ruhe, Germany) with 20 mm diameter parallel serrated plates probe ture of 100 ◦ C was increased at 8 ◦ C/min to 180 ◦ C and held at this with 2 mm gap at 4 ◦ C. One spoonful of the sample was carefully loaded temperature for 9 min and further increased at 1 ◦ C/min to 230 ◦ C and onto the plate of the rheometer, and once the measure position was held at this temperature for 10 min (total run time: 84 min). Detector gas reached, the sample was left to stand for 5 min before the test started. flow: H2, 40 mL/min; make-up gas (He), 25 mL/min; air, 400 mL/min. Frequency sweep was performed in the range of 0.1–10 Hz, in the viscoelastic linear region previously determined. The viscoelastic pa­ 2.8. Microbiological analysis rameters, storage modulus (G′) and loss modulus (G’’) were recorded. Confocal laser scanning microscopy (CLSM) observations were per­ Mesophilic aerobic bacteria, aerobic spores, coliform bacteria, and formed in a Leica TCS SP2 AOBS microscopy (Leica, Heidelberg, Ger­ E. coli were determined on RM samples. Samples were serially diluted in many). The protein matrix was stained using 10 mg of fluorescein peptone water (Oxoid, Basingstoke, UK) and plated on Plate Count Agar isothiocyanate (FITC) (Fluka, Sigma- Aldrich, Germany) completely (PCA, Oxoid) to enumerate mesophilic aerobic bacteria. Viable counts of dissolved in ethanol. Then, it was added to 5 mL of preheated and spores were enumerated after the serial dilutions of samples were heated inoculated RMs. After incubation at 43 ◦ C for 4 h, yogurts were kept at 80 ◦ C for 10 min, quickly cooled, poured into PCA plates, and incu­ overnight at refrigeration temperature and further stirred. Microscope bated at 30 ◦ C for 48 h. For the enumeration of coliforms/E. coli, serial observations were made with × 63 oil immersion objective at wave­ dilutions were plated on the surface of Chromogenic Coliform Agar length of 488 nm and with 1024 × 1024 format. (CCA) and incubated at 37 ◦ C for 24 h. Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) and Streptococcus thermophilus were enumerated 2.5. Physicochemical characteristics of yogurts: color, pH, total acidity, according to ISO 7889:2003. For each treatment, samples were analyzed and water holding capacity (WHC) during storage at 4 ◦ C on days 1, 14 and 28. Yogurt samples were serially diluted from 10 − 1 to 10 − 6 in peptone water. L. bulgaricus and The color of yogurts was assessed with a colorimeter Konica Minolta S. thermophilus were counted in the selective media MRS and M17, under CR-410 (Konica Minolta, Osaka, Japan), with a reference D65 light anaerobic conditions, at 37 ◦ C for 72 h and 37 ◦ C for 48 h, respectively. source and a 10◦ observer angle. The CIE L* a* and b* color coordinates All samples were analyzed in duplicate. were obtained. The following formulas were utilized to determine the Yellowness Index (YI) and Whiteness Index (WI), respectively. 2.9. Sensory evaluation A panel of 20 university faculty and staff members who were familiar 1 YI = 142.86 × b* × L*− 2 2 0.5 with yogurt (mean age = 31 years, with an age range between 22 and 61 WI = 100 − [(100 – L*) + a* + b* ] years), were asked to identify differences. The sensory evaluation con­ The pH of yogurts was measured using Crison Basic 20 pH-meter sisted of triangle, descriptive, and preference tests. Two triangle tests (Crison Instruments S.A., Alella, Spain). Titratable acidity (TA) was were carried out on days 7 and 14 after yogurt manufacture to assess determined according to the ISO/TS 11869:2012 method. whether the judges could find significant differences between the two The WHC was determined by centrifuging 40 g of yogurt at 5000 g for treatments of CH4 vs UH4 and CH6 vs UH6 or not. Once the results of the 20 min at 22 ◦ C to separate the supernatant (whey) in triplicate on days first triangle test had been analyzed, the effect of the concentration 1, 14, and 28. The following equation was used to calculate WHC g/100 incorporated into the yogurts (UH4 vs UH6) was compared using g: another triangle test on day 14. A descriptive test based on a 7-point intensity scale (1: dislike extremely; 7: like extremely) was conducted WHC g/100 g = (W1 – W2)/W1 × 100 to identify the different attributes (creaminess, consistency, acidity, Where: W1 =Initial yogurt weight, W2 = Weight of whey after strange aroma, and lactic aroma) in the stirred yogurt. Finally, the centrifugation. judges were asked to rank the samples of yogurt between treatments UH4 and UH6 in order of preference. 2.6. Stability to oxidation 2.10. Statistical analysis Quantification of hydroperoxides and malondialdehyde in yogurts Effects of different levels of SDE (treated with CH and UHPH) addi­ was performed by the methods proposed respectively by Hu, Julian, and tion, and storage time on physical, chemical, and sensory parameters Decker (2003) and Papastergiadis, Mubiru, Van Langenhove, and De were analyzed based on one-way and two-way analysis of variance Meulenaer (2012) which were carried out with slight modification and (ANOVA) test using the Minitab Express™ version 1.5.3 (Minitab, State were fully described previously (Varela et al., 2022). College, PA, USA). Significant differences between means were deter­ mined by the Tukey test. A confidence level of 95% (P < 0.05) was used. 2.7. Fatty acid composition of yogurts At least three individual productions of each formulation and treatment were performed. Oleic, linoleic, and α-linolenic acid content of yogurts fortified with 4 and 6 g/100 g of SDE was determined on days 1 and 28 of storage by 3. Results and discussion using a gas chromatograph HP-6890 Series GC System (Hewlett-Pack­ ard, Waldbronn, Germany) equipped with a flame ionization detector 3.1. Acid coagulation process and a HP-6890 Series Injector. A capillary column VF-5 ms (Agilent Technologies, California, U. S.), 30 m × 0.25 mm with 0.25 μm film During the acidification process of yogurt, the milk caseins become thickness containing 5% phenyl-methylpolysiloxane was used. The unstable and coagulate, forming a protein matrix that entraps the 3 F. Aghababaei et al. LWT 187 (2023) 115304 aqueous phase and oil droplets, with varying degrees of interaction between them (Alexander & Dalgleish, 2004). Table 1 shows the acid coagulation parameters of samples incubated at 43 ◦ C. Evolution of pH during the coagulation process is depicted in Fig. 2. The onset of gelation (OG) occurred significantly earlier in UHPH- RMs compared to CH-RMs. Additionally, increasing the percentage of added SDE (spray dried emulsion) resulted in earlier OG. These results could be attributed to the smaller size of oil droplet aggregates in UHPH- RMs, which had a larger effective surface area compared to the indi­ vidual oil droplets in CH-RMs (Varela et al., 2022). The smaller aggre­ gates in UHPH-RMs contribute to the earlier onset of gelation, as observed in previous studies (Serra, Trujillo, Quevedo, Guamis, & Fer­ ragut, 2007). However, once gelation started, the aggregation rate (AR) and the final gel firmness (FGF) were both higher in CH-RMs. In previous studies (Aghababaei et al., 2021; Varela et al., 2022), liquid emulsions Fig. 2. pH curves as a function of time during fermentation of the different treated by the two different homogenization systems showed different types of yogurts. Green: CH4, Yellow: CH6, Purple UH4, and Blue: UH6. morphology of colloidal structures. The colloidal structures from CH treatments presented larger spherical droplets, whereas those treated similarities and differences emerged between the yogurts during stor­ with UHPH, especially those treated at 200 MPa, presented a greater age. CH6 and UH4 yogurts appeared to exhibit similar firmness and number of asymmetric aggregates of smaller size than the previous ones. consistency, suggesting that the flow during the compression of the gel It is to be expected that when reconstituting the SDEs to form the RMs, resulted in the formation of yogurt flocs of similar size. However, UH6 these colloidal structures were maintained to a greater or lesser extent. yogurts showed the lowest values of both firmness and consistency. In yogurt, it is known that the predominant interactions in gel formation Although it may seem contradictory that increasing SDE concentration are of the casein-casein type, so it could be hypothesized that the higher in UHPH yogurts displayed the lowest texture parameter values, this AR and FGF observed in CH yogurts could be due to the predominance of could be explained by the previous studies performed in the emulsions these interactions, since there would be more continuity of casein for and SDE with the same compositions as in the present study (Varela their interaction. In contrast, in UHPH yogurts, the greater amount of et al., 2022). In this study, the oil was present in the UHPH-treated particle aggregates would act as disruptors of the continuity of these emulsions in the form of aggregated droplets through proteins. These interactions. colloidal structures in higher concentrations in UH6 yogurts could act as The acidification curves of the yogurts did not show significant dif­ disruptive particles, interrupting the continuity of the casein network. ferences between treatments, indicating similar kinetics of fermentation As a result, more open spaces were present in the stirred yogurts, as (P < 0.05). The overlapping acidification curves in Fig. 2 demonstrate depicted in Fig. 3, which ultimately led to reduced firmness and con­ that pH 4.6 was reached after approximately 198 ± 3 min. This aligns sistency of those yogurts. Ciron et al. (2010) in low-fat stirred yogurts with the findings of previous studies, which reported incubation times of also described the formation of fat globule aggregates when comparing about 183–240 min to reach the desired pH (Horiuchi et al., 2009; conventional homogenization and microfluidization at 150 MPa. Soukoulis, Panagiotidis, Koureli, & Tzia, 2007). Microscopic observations revealed that microfluidized milk-based yo­ gurts exhibited more open spaces compared to those made with conventionally treated milk, impacting the texture parameters obtained 3.2. Texture, rheology, and microstructure Texture parameters, specifically firmness and consistency, of the yogurts were assessed using back extrusion testing during storage at 4 ◦ C. Firmness refers to the maximum force applied during compression, while consistency indicates the thickness of the sample (Ciron, Gee, Kelly, & Auty, 2010). Back extrusion testing provides insights into the behavior of the sample as it flows back under applied compression, leading to the destruction of the gel microstructure. On the first day, there were no significant (P > 0.05) differences in texture parameters between the various yogurt samples. At this early stage, when the stirred gel has not settled yet, the main factor influ­ encing yogurt texture is likely the crystallized fat under refrigerated conditions (Serra et al., 2007), which was consistent across all yogurts. However, as the gel structure settled on days 14 and 28, some Table 1 Coagulation parameters of yogurts containing different content (4 and 6%) of CH and UH SDE. 1) Sample OG (min) AR (mA min− FGF (mA) a b CH4 115±1 0.35 ± 0.01 41.3 ± 0.4b CH6 110.4 ± 0.2b 0.372 ± 0.001a 45.3 ± 0.3a UH4 106.5 ± 0.3c 0.332 ± 0.001c 37.8 ± 0.5c UH6 102±1d 0.346 ± 0.003b 40.5 ± 0.4b Means in each column with different superscript letters were significantly different (P < 0.05). OG: onset of gelation; AG: aggregation rate; FGF: gel firmness at the end of Fig. 3. CLSM images of the different types of yogurts: CH4 (A), CH6 (B), UH4 gelation (n = 3). (C) and UH6 (D). 4 F. Aghababaei et al. LWT 187 (2023) 115304 through back extrusion testing. Table 3 Dynamic oscillatory testing with small amplitudes within the linear Color parameters (WI, YI), WHC, TA and pH values of different yogurt samples viscoelastic region allowed the assessment of interaction strength during storage at 4 ◦ C. responsible for the network formation in semi-solid and solid foods Sample/days 1 14 28 without disrupting their structure. G’ (storage modulus) represents the WI intensity and/or number of interactions contributing to network for­ CH4 94.86 ± 0.01ab,B 94.89 ± 0.03a,A 94.81 ± 0.01b,B mation, while G’’ (loss modulus) relates to the viscous nature and in­ CH6 94.81 ± 0.01b,B 94.72 ± 0.01c,C 94.916 ± 0.005a,A teractions that do not directly affect the three-dimensional network UH4 94.74 ± 0.01b,C 94.776 ± 0.004a,B 94.68 ± 0.01c,C UH6 94.50 ± 0.01b,D 94.488 ± 0.005b,D 94.64 ± 0.02a,D (Tabilo-Munizaga & Barbosa-Cánovas, 2005). All yogurts exhibited YI characteristic solid behavior, with higher G′ values than G’’ values, as CH4 4.68 ± 0.05a,D 4.45 ± 0.06b,D 4.39 ± 0.07b,B shown in Table 2. CH yogurts consistently displayed significantly higher CH6 4.83 ± 0.04b,C 4.91 ± 0.01a,C 4.365 ± 0.009c,B (P < 0.05) G′ values than UHPH yogurts throughout the storage period. UH4 5.05 ± 0.02a,B 5.018 ± 0.007b,B 5.08 ± 0.01a,A Moreover, the increase in viscoelastic parameters with the addition of UH6 5.68 ± 0.03b,A 5.85 ± 0.01a,A 5.14 ± 0.05c,A pH higher percentages of SDE aligned with this difference, as expected. CH4 4.50 ± 0.04a,A 4.30 ± 0.02b,A 4.25 ± 0.01b,A Furthermore, all yogurts showed a significant increase in both G′ and G’’ CH6 4.47 ± 0.06a,A 4.29 ± 0.01b,A 4.23 ± 0.01b,A from day 1 to day 28 of cold storage, indicating the settling of the gel UH4 4.52 ± 0.01a,A 4.28 ± 0.03b,A 4.226 ± 0.005c,A structure produced by increased protein-protein interactions, favored, UH6 4.52 ± 0.01a,A 4.30 ± 0.03b,A 4.23 ± 0.02b,A TA (mmol/100g) among other factors, by pH decreasing over storage. CH4 16.6 ± 0.1b,A 17.1 ± 0.6ab,A 17.5 ± 0.2a,A CH6 16.8 ± 0.1a,A 17.1 ± 0.2a,A 17.1 ± 0.2a,AB UH4 16.01 ± 0.07c,B 16.7 ± 0.2b,A 17.0 ± 0.1a,AB 3.3. Evaluation of color, pH, acidity and WHC UH6 15.81 ± 0.07b,B 16.4 ± 0.4a,A 16.6 ± 0.3a,B WHC (%) CH4 31.0 ± 0.2b,C 31.1 ± 0.1b,D 31.8 ± 0.3a,C Consumer preference is influenced by the immediate perception of CH6 35.20 ± 0.07a,A 35.2 ± 0.4a,B 35.9 ± 0.7a,A color, which is a fundamental characteristic. The color coordinates (L*, UH4 33.1 ± 0.1a,B 33.1 ± 0.3a,C 33.5 ± 0.4a,B a*, b*) of the yogurts were measured during storage, with L* values UH6 35.7 ± 0.5a.A 36.0 ± 0.1a,A 36.3 ± 0.6a,A representing luminosity, the white achromatic attribute. L* values for all Different capital letters in each column indicate significant differences (P < yogurts and storage periods were similar, ranging from 82.9 to 83.9. 0.05). Different small letters in each row indicate significant differences. Stirred yogurt, characterized by a rough surface due to the gel particles (P < 0.05). Each value is expressed as mean ± SD (n = 3). formed during beating, exhibited a slightly lower diffuse reflection than expected. However, neither the type nor the content of SDE added to the yogurt samples. A significant decrease (P < 0.05) in pH was observed RM had a significant influence on luminosity. The a* (green-red) and b* from day 1 to day 14 of storage due to the high metabolic activity of the (blue-yellow) coordinates indicated positive values, with red and yellow yogurt starters during the initial stages. The pH remained constant from contributing to the chromatic component of the color, respectively. The day 14 until day 28 when yogurt production ceased in refrigeration a* values ranged from 1.51 to 1.83, while the b* values ranged from 2.55 (Lucey, 2004). to 3.34, with the latter indicating a predominant yellow contribution to The titratable or total acidity of yogurts should ideally be within the the yogurt color. The calculated values of whiteness index (WI) and range of 0.5–1.6 g/100 mL according to quality standards for fermented yellowness index (YI) (Table 3) demonstrated the prevalence of white­ milks (WHO/FAO, 2003). Table 3 displays the total acidity values for ness over yellowness, with a slightly higher yellowness observed in each type of yogurt during the 28-day storage at 4 ◦ C. Initially, CH yo­ UHPH yogurts. gurts exhibited higher total acidity compared to UHPH yogurts. This There were no significant differences in pH values between any of the initial difference persisted during storage, although it was not signifi­ cantly different (P > 0.05) from day 14 onwards. A study by Serra, Table 2 Trujillo, Guamis, and Ferragut (2009) on physicochemical characteris­ Mean values ± SD of textural and rheological parameters of yogurts during tics of yogurts made from CH and UHPH-treated milks also reported storage at 4 ◦ C. higher total acidity in CH yogurts during storage. In the present study, Sample d1 d14 d28 lactose primarily derived from UHT milk and skim milk powder used in Firmness (N) RM formulations remained constant across all yogurts. Therefore, there CH4 0.23 ± 0.04a,A 0.214 ± 0.004ab,B 0.183 ± 0.003b,C were no significant differences (P > 0.05) in total acidity between yo­ CH6 0.24 ± 0.03a,A 0.22 ± 0.01ab,AB 0.200 ± 0.005b,B gurts with 4 g/100 mL or 6 g/100 mL added SDE. Furthermore, differ­ UH4 0.26 ± 0.03a,A 0.233 ± 0.007a,A 0.22 ± 0.01a,A UH6 0.21 ± 0.02a,A 0.190 ± 0.008b,C 0.183 ± 0.004b,C ences in the perceived sensory acidity between the samples was deemed Consistency (N*s) insignificant, as will be shown in the corresponding section. It is worth CH4 0.9 ± 0.1a,A 0.82 ± 0.02a,AB 0.78 ± 0.01a,B noting that the total acidity in this study was within the higher end of the CH6 0.9 ± 0.1a,A 0.84 ± 0.04a,A 0.80 ± 0.01a,AB recommended range. This may be attributed to an over-acidification UH4 1.0 ± 0.1a,A 0.87 ± 0.03a,A 0.85 ± 0.03a,A experienced on day 0, as the fermentation process was stopped below UH6 0.8 ± 0.1a,A 0.77 ± 0.01a,B 0.76 ± 0.03a,B G’ (Pa) pH 4.6. CH4 67±1c,B 99±1b,B 128.5 ± 0.5a,B Syneresis, the expulsion of the aqueous phase from the yogurt gel, is CH6 87.1 ± 0.9c,A 120.9 ± 0.8b,A 158±1a,A a common issue that affects product quality (Lucey, 2004). The amount UH4 60.7 ± 0.9c,D 66±1b,D 97.2 ± 0.5a,D of expelled aqueous phase is inversely related to water holding capacity UH6 65±1c,C 86.8 ± 0.08b,C 100±1a,C G’’ (Pa) (WHC). Hence, greater expulsion results in lower WHC. To minimize CH4 18.7 ± 0.6c,B 25.7 ± 0.3b,B 33±1a,B syneresis, skim milk powder (SMP) and/or milk proteins are commonly CH6 24,7 ± 0.2c,A 32.0 ± 0.5b,A 40±1a,A added in yogurt production. In this study, 3 g/100 g SMP was added to UH4 17.2 ± 0.4b,C 17.4 ± 0.3b,D 26±1a,C all RMs. Centrifugation was used to induce syneresis and compare the UH6 18.6 ± 0.3c,B 22.8 ± 0.2b,C 26.1 ± 0.3a,C WHC of the different yogurt types produced. It is important to note that Different capital letters in each column indicate significant differences (P < centrifugation-induced syneresis does not fully represent the real 0.05). network contraction of yogurts but rather indicates the gel structure Different small letters in each row indicate significant differences (P < 0.05) (n ability to retain water. Results of WHC (Table 3) showed that CH6 and = 3). 5 F. Aghababaei et al. LWT 187 (2023) 115304 UH6 yogurts had significantly (P < 0.05) higher WHC compared to CH4 both on days 1 and 28. As was observed in 7SDE by Varela et al. and UH4 yogurts. Furthermore, UHPH yogurts exhibited significantly (2022), the encapsulation efficiency of UHPH-SDE, in general and, in higher WHC compared to CH yogurts. Thus, in this study, the SDE 7UHPH-SDE, in particular, were higher than those SDE obtained by CH. content and UHPH treatment were responsible for increasing water Probably, the small droplets which were well covered by phospholipids retention in yogurts. The positive contribution of UHPH treatment may and proteins forming aggregates were responsible for keeping the oil be attributed to the higher water retention in the interior and at the well retained in those colloidal structures, and therefore, protecting oil interface of the oil-protein aggregates in UHPH-treated yogurts, as against oxidation. observed by Varela et al. (2022) in SDE obtained through UHPH treat­ ment. It has been reported that acid-induced gelation of protein-based emulsions with high fat content leads to higher WHC (Gyawali & Ibra­ 3.5. The composition of the main unsaturated fatty acids him, 2016). This could be due to the oil droplets, bound by proteins at the oil-water interface, acting as protein particles in the case of UHPH The main composition of unsaturated fatty acids (oleic, linoleic, and SDE, thereby increasing immobilized water. Moreover, the WHC α-linolenic acids) in yogurts on days 1 and 28 of storage, is presented in remained stable during the storage of each sample. Although water Table 4. Linoleic acid was found to be the most abundant poly­ expulsion is a time-dependent phenomenon due to progressive unsaturated fatty acid (PUFA) in all yogurts due to the 50:50 ratio of compaction of the yogurt network over time (Li, Ye, & Singh, 2021; chia and sunflower oils in their composition. This oil mixture was Gilbert & Turgeon, 2021), the values obtained through centrifugation selected to mitigate the strong green aroma of pure chia oil and also included water expelled by spontaneous syneresis, which was contribute to a balanced ratio of omega-6 to omega-3 fatty acids in observed in small quantities and could not be measured accurately. yogurts. Sunflower oil is rich in oleic and linoleic fatty acids, while chia oil primarily contributes to the α-linolenic acid content, and to a lesser extent the linoleic acid content, of the mixture. As anticipated, 3.4. Stability to oxidation increasing the amount of SDE added to RMs resulted in higher levels of fatty acids in yogurts. In all cases, UHPH treatment significantly Chia and sunflower oils are rich in PUFA, thus they are prone to increased the content of unsaturated fatty acids in yogurts compared to oxidation during processing and storage, which may produce off-flavors CH. UHPH yogurts contained higher amounts of oleic, linoleic, and (Estrada, Boeneke, Bechtel, & Sathivel, 2011). Oxidation of yogurts α-linolenic acids. As mentioned earlier (Aghababaei et al., 2021; Varela containing chia and sunflower oils (50:50) were evaluated for primary et al., 2022), the encapsulation efficiency of UHPH-treated SDE was (hydroperoxide concentration) and secondary oxidation (malondialde­ higher than that of CH, providing better protection against oxidation, as hyde, MDA, concentration) at days 1 and 28. The effect of type of ho­ mogenization system to obtain SDE and the percentage added to RMs (4 and 6 g/100 g) on the primary and secondary oxidation of yogurts is Table 4 Fatty acid composition (mg/100g) of the different yogurts on days 1 and 28 of shown in Fig. 4. Hydroperoxide concentration (Fig. 4A) of CH yogurts storage at 4 ◦ C. was significantly higher (P < 0.05) than UHPH yogurts on day 1 as well as on day 28 of storage. Moreover, the increase of those values of Sample/ Oleic acid Linoleic acid Linolenic acid FFAA UHPH-yogurts from day 1 to day 28 was much less pronounced than d1 d28 d1 d28 d1 d28 those observed in CH yogurts. Varela et al. (2022) observed similar re­ CH4 107.80 84.16 ± 201.92 146.89 190.01 112.30 sults in SDE containing 7 g/100 g BM, like in the present study, i. e. ± 3.87a, 4.36b,B ± 2.99a,C ± ± 3.12a, ± 3.74b,D 7CH-SDE was more prone to primary oxidation than 7UHPH-SDE. B 2.03b,C C However, they performed the study of oxidation stability in forced CH6 185.61 149.53 256.26 233.84 227.73 174.80 ± 5.73a, ± 7.85b,A ± 8.29a,A ± 4.10a, ± 3.64b,B conditions at 50 ◦ C for one month and observed a maximum value of ± A 2.34b,A A hydroperoxide concentration at day 15, which was followed by a UH4 118.61 94.47 ± 216.77 189.86 210.79 158.66 reduction at day 31, meaning that the secondary oxidation was in ± 6.81a, 3.16b,B ± 2.97a,B ± ± 8.08a, ± 1.99b,C B progress by the degradation of hydroperoxides. In the present study, the 3.81b,B B UH6 194.91 155.463 268.167 242.23 240.73 193.573 oxidation was analyzed in yogurts at 4 ◦ C, thus the further degradation ± 2.05a, ± 1.420b, ± 1.419a, ± ± 4.94a, ± 1.615b, of hydroperoxides was not produced during the storage. A A A 6.38b,A A A One of the final products of polyunsaturated fatty acid oxidation is Different capital letters in each column indicate significant differences for each MDA, which is indicative of secondary oxidation (Fig. 4B). With the sample and for each FA in the same day (P < 0.05). exception of UH4, which on day 1 of storage showed higher MDA values Different lowercase in each row for each FA indicate significant differences for than CH4, this parameter showed a resembling behavior as primary each sample in days 1 and 28 (P < 0.05) (n = 3). oxidation, with higher MDA values in CH compared to UHPH-yogurts, Fig. 4. Hydroperoxides (A) and malondialdehyde (B) concentration of yogurts at days 1 and 28 of storage at 4 ◦ C. Purple: CH4, Green: CH6, Yellow: UH4, Blue: UH6. 6 F. Aghababaei et al. LWT 187 (2023) 115304 observed in section 3.4. During storage, a significant decrease in FA percentages of SDE (4 g/100 g or 6 g/100 g) and the type of homoge­ concentrations was observed in yogurts. In addition to the high sus­ nization (CH or UHPH) were detectable in the yogurts. The sets of tri­ ceptibility of unsaturated FA to oxidation, hydrolysis of triglycerides angle tests were carried out sequentially, comparing two different with the liberation FA, which was observed in the over-acidification samples in each set, based on the results of the previous tests. The first during storage, may have also contributed to the oxidation (Table 3). set of triangle testing (Fig. 5A) compared CH4 vs UH4 yogurts, and no Despite the reduction in FA levels during storage, the PUFA content of significant differences were detected. Therefore, the next set of triangle yogurts makes a valuable contribution to the diet. Yogurt is a food tests (Fig. 5B) compared CH6 vs UH6 yogurts, and again, no significant consumed daily by many people and the intake of these essential fatty differences were observed. Subsequently, a third set of triangle tests acids contributes to a balanced diet. (Fig. 5C) compared UH4 vs UH6 yogurts, based on the superior quality characteristics observed in this study compared to CH yogurts. The re­ 3.6. Microbiology sults of this set of triangle testing did not show any significant differ­ ences (P < 0.05) between UH4 and UH6, indicating that none of the Initial microbial counts of RMs for further production of yogurts pairs of samples were distinguished by the judges. This is particularly were performed in selective media for coliforms/E. coli, mesophilic important because the addition of 6 g/100 g SDE, which contains a aerobes, and their spores. No coliform/E. coli colonies were detected in higher concentration of PUFA, did not result in any unusual flavors in any of the samples, and, in the case of mesophilic aerobes and their the yogurts. spores, the mean values were 1.70 and 1.34 log CFU/mL, respectively. For the descriptive test, five parameters associated with the sensory European legislation (WHO/FAO, 2003) states that the lactic acid profile of the yogurts were evaluated: consistency, creaminess, acidity, bacteria in yogurt (L. bulgaricus and S. thermophilus) must be viable and lactic aroma, and off-flavors. Fig. 5D illustrates that UH6 obtained present in the finished product in a minimum amount of 1 × 107 CFU/g higher scores than UH4 in all parameters except "lactic aroma" and or mL. Table 5 shows the microbial counts of each microorganism in "acidity." "Consistency" and "creaminess" were the only parameters that yogurts after 0, 1, 14, and 28 days in MRS and M17 media (selective for showed a significant difference (P < 0.05) between the two samples. L. bulgaricus and S. thermophilus, respectively). Counts after yogurt UH6 yogurts obtained scores of 4.3 ± 0.4 for consistency and 4.43 ± production corresponded to the RM inoculated and analyzed prior to the 0.43 for creaminess on a scale of 0–5, while UH4 yogurts obtained scores fermentation process, which explains the significant difference between of 3.8 ± 0.4 and 3.75 ± 0.35, respectively, for those attributes. Addi­ the counts on day 0 and those on subsequent days. Once the fermenta­ tionally, although not significantly different (P > 0.05), the preference tion process was completed, bacterial growth was, in general, around 7 test conducted between UH4 and UH6 showed that yogurts with 6 g/ log CFU/g, as required by legislation. Yogurts made with SDE treated by 100 g SDE received higher scores, likely due to the better texture CH or UHPH showed no significant differences (P > 0.05) between them perceived in the descriptive test. and no effect related to the percentage of SDE added to yogurts was observed. Counts of L. bulgaricus remained constant during the storage of 4. Conclusions yogurts while S. thermophilus showed a significant increase (P < 0.05) in counts on day 28, which could be related to a greater adaptation of The choice of homogenization system (CH or UHPH) influenced the S. thermophilus to the formulations used. quality parameters of the yogurts. CH treatment produced yogurts with higher firmness and consistency compared to UHPH treatment. How­ ever, UHPH yogurts improved WHC and offered better protection 3.7. Sensory evaluation against oxidation, preserving PUFA content during storage compared to CH yogurts. Importantly, sensory evaluation indicated that neither the Sensory evaluations were conducted using triangle testing, followed homogenization system nor the SDE content significantly impacted by descriptive and preference testing. Twenty judges participated in yogurt flavor, allowing for the use of higher SDE concentrations without different sets of triangle tests to determine if the addition of different altering sensory attributes. UHPH technology, with its ability to simul­ taneously homogenize and modify colloidal structures, has the potential Table 5 for broad applications in the food industry. It offers opportunities for Microbiological counts (mean log CFU/g ± SD) of recombined milks inoculated improved emulsion stability and interaction between emulsifiers and the with yogurt starters (day 0) and yogurt samples during storage at 4 ◦ C (days 1, oil phase. 14 and 28). Sample/ d0 d1 d14 d28 Statement days L. bulgaricus During the preparation of this work the authors used Chatgpt in CH4 4.56 ± 0.22b, 7.01 ± 0.62ab, 7.85 ± 1.74a, 7.53 ± 0.49a, order to improve language and readability. After using this tool, the A A A A authors reviewed and edited the content as needed and take full re­ CH6 4.60 ± 0.30b, 6.91 ± 0.42a,A 6.96 ± 1.50a, 7.28 ± 0.56a, A A A sponsibility for the content of the publication. UH4 4.55 ± 0.23b, 7.24 ± 0.30a,A 6.78 ± 0.81a, 7.41 ± 0.70a, A A A CRediT authorship contribution statement UH6 4.58 ± 0.21b, 7.36 ± 0.40a,A 6.81 ± 0.86a, 7.45 ± 0.50a, A A A S. thermophilus Fatemeh Aghababaei: Investigation, Writing – original draft. CH4 4.17 ± 1.63b, 8.50 ± 0.71a,A 8.44 ± 0.96a, 9.15 ± 0.00a, Antonio J. Trujillo: Validation, Project administration. Bibiana Juan: A A B Validation, Methodology, Data curation. Marta Capellas: Validation, CH6 3.69 ± 0.65b, 8.58 ± 0.65a,A 8.41 ± 0.96a, 9.19 ± 0.00a, Methodology. Victoria Ferragut: Conceptualization, Methodology, A A A Data curation, Writing – review & editing, Supervision. UH4 3.17 ± 0.39b, 8.79 ± 0.47a,A 8.51 ± 0.98a, 9.14 ± 0.00a, A A C UH6 3.79 ± 0.59b, 8.58 ± 0.67a,A 8.46 ± 0.92a, 9.13 ± 0.00a, Declaration of competing interest A A C Different capital letters in each column indicate significant differences (P < The authors declare that they have no known competing financial 0.05). Different small letters in each row indicate significant differences (P < interests or personal relationships that could have appeared to influence 0.05) (n = 2). the work reported in this paper. 7 F. Aghababaei et al. LWT 187 (2023) 115304 Fig. 5. Sensory analysis of yogurts. Triangular testing of the different pairs of yogurts: CH4 vs UH4 (A), CH6 vs UH6 (B), UH4 vs UH6 (C). Descriptive test of yogurts UH4 (green line) and UH6 (purple line) (D). Data availability Donovan, S. M., & Rao, G. (2019). 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